枣黑斑病菌细胞壁降解酶活性测定及致病性分析
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摘要
【目的】以枣黑斑病菌产生的细胞壁降解酶为研究对象,明确其产生细胞壁降解酶种类和活性,探讨细胞壁降解酶在病菌致病中的作用,为细胞壁降解酶参与病原菌侵染机理的研究提供理论依据。【方法】利用3,5-二硝基水杨酸(DNS)法和考马斯亮蓝(Bradford)法,通过紫外-可见分光光度计测定反应混合物吸光度,根据酶反应所释放的还原糖计算细胞壁降解酶活性。【结果】枣黑斑病菌在寄主体外不同碳源诱导下均能产生多聚半乳糖醛酸酶(Polygalacturonase, PG)、羧甲基纤维素酶(Carboxymethyl cellulase, Cx)、β-葡萄糖苷酶(β-glucosidase)、木聚糖酶(Xylanase)、聚甲基半乳糖醛酸酶(Polymethylgalacturonase, PMG)和果胶甲基反式消除酶(Pectin methyltranseliminase, PMTE)等6种细胞壁降解酶(Cell wall degrading enzyme,CWDE),但细胞壁降解酶活性存在一定差异,以骏枣果肉为外源诱导物,枣黑斑病菌产生β-葡萄糖苷酶活性明显高于其他5种酶,说明β-葡萄糖苷酶在枣黑斑病菌致病过程中有着重要的作用,而以滤纸为诱导物产生的β-葡萄糖苷酶活性高于其他诱导物,其活性高达10.104 U·mg~(-1)。枣黑斑病菌侵染枣果后产生的细胞壁降解酶种类及活性与体外不同碳源诱导的结果一致,可产生6种细胞壁降解酶,其中β-葡萄糖苷酶活性高于其他5种酶,且病健交界处β-葡萄糖苷酶的活性明显高于受侵染后的发病部位和未被侵染的部位。【结论】枣黑斑病菌在致病过程中起关键作用的细胞壁降解酶为β-葡萄糖苷酶,且在侵染过程中病健交界处的活性最高,而在寄主体外诱导β-葡萄糖苷酶最佳碳源为滤纸诱导物。
【Objective】The cell wall degrading enzymes produced by the black spot pathogen were studied, and their activities were determined. The role of cell wall degrading enzymes in the pathogenesis of the pathogens was discussed.【Methods】Jujube black spot disease was isolated from jujube in southern Xinjiang. Pathogen hyphae were separated from PDA culture medium. The hyphea were added into induction media containing sucrose, pectin, cellulose, filter paper powder, cotton powder, or jujube pulp as the inducers. The media were shaken in a shaker incubator set at 25 ℃ for 7 days, vacuum filtered to remove hyphae and spores, and centrifuged at 4 ℃ for 30 minutes at 10 000 r·min~(-1). The supernatant was used as the crude enzyme solution. The crude enzyme solution was vacuum filtered, and ethylenediamine tetraacetic acid was added to the filtrate. Then ammonium sulfate was added slowly with gentle stirring until 60% saturation. The mixture was allowed to stand at 4 ℃ for 5 hours and centrifuged at4 ℃ 15 000 r·min~(-1) for 20 minutes. The supernatant was discarded, and the precipitation was dissolved with acetic acid-sodium acetate buffer and placed in a dialysis bag. In the same buffer, dialysis was carried out at 4 ℃ for 48 hours and the dialysate was changed every 12 hours. The purified enzyme was used for enzyme activity determination. Crude enzymes were also extracted in vivo from different parts of jujube diseased fruit inoculated with spore suspension. The tissues were cut into slices and put into mortar, grinded with sodium chloride at 4 ℃, centrifuged at 4 ℃ and 5 000 r·min~(-1) after filtration, and the supernatant was collected as the crude enzyme for later use. Activities of six cell wall degrading enzymes were measured using the 3, 5-dinitrosalicylic acid(DNS) method and the Coomassie blue staining method. Among them, the activities of polygalacturonase, carboxymethylcellulase, β-glucosidase,xylanase and polygalacturonase were measured by the 3, 5-dinitrosalicylic acid method using pectin,carboxymethyl cellulose, salicin and xylan as substrate, respectively. Substrate solution was prepared with citric acid buffer. Using citric acid buffer solution as the blank, the enzymic hydrolysis was carried out at 50 ℃ for 30 to 60 minutes. The absorbance of the reaction mixture was measured by UV-Vis spectrophotometer, and the the enzyme activities were calculated according to the reducing sugar released. The pectin methyl trans-eliminating enzyme was determined by Coomassie Brilliant Blue method. The color density of the protein-Coomassie brilliant blue complex solution was proportional to protein concentration. The differences in color of the solution created by enzymic reactions was used to calculate the cell wall degrading enzyme activity.【Results】Jujube black spot bacteria produced six cell wall degrading enzymes under the induction of different carbon sources. They were polygalacturonase,carboxymethylcellulase, β-glucosidase, xylanase, polymethylgalacturonase and pectin methyl transeliminase. However, there were some differences in activity among enzymes under different inducers.When the jujube pulp was used as the inducer, the activity of β-glucosidase produced by the black spot pathogen was significantly higher than that of the other five enzymes, indicating that β-glucosidase played an important role in the pathogenesis of black spot pathogen. Filter paper as the inducer also produced the highest β-glucosidase activity. The activities of cell wall degrading enzymes produced by jujube black spot in infected jujube fruit was consistent with the results induced by different carbon sources in vitro, with the activity of β-glucosidase being higher than the other five enzymes. Moreover, the activity of β-glucosidase at the junction of diseased and healthy parts was the highest among different tissues sites.【Conclusion】Different inducers have different effects on the cell wall degrading enzymes secreted by the black spot pathogen, and the inducer most favorable for pathogen growth has an important effect on the enzyme production. Filter paper can be used as a good inducer of β-glucosidase. Combining the enzyme activity assay results in vitro and in vivo, it is speculated that β-glucosidase is the major cell wall degrading enzymes that play a role in the pathogenesis of the black spot pathogen. The key part of the impact is at the edge of the diseased tissue.
【Objective】The cell wall degrading enzymes produced by the black spot pathogen were studied, and their activities were determined. The role of cell wall degrading enzymes in the pathogenesis of the pathogens was discussed.【Methods】Jujube black spot disease was isolated from jujube in southern Xinjiang. Pathogen hyphae were separated from PDA culture medium. The hyphea were added into induction media containing sucrose, pectin, cellulose, filter paper powder, cotton powder, or jujube pulp as the inducers. The media were shaken in a shaker incubator set at 25 ℃ for 7 days, vacuum filtered to remove hyphae and spores, and centrifuged at 4 ℃ for 30 minutes at 10 000 r·min~(-1). The supernatant was used as the crude enzyme solution. The crude enzyme solution was vacuum filtered, and ethylenediamine tetraacetic acid was added to the filtrate. Then ammonium sulfate was added slowly with gentle stirring until 60% saturation. The mixture was allowed to stand at 4 ℃ for 5 hours and centrifuged at4 ℃ 15 000 r·min~(-1) for 20 minutes. The supernatant was discarded, and the precipitation was dissolved with acetic acid-sodium acetate buffer and placed in a dialysis bag. In the same buffer, dialysis was carried out at 4 ℃ for 48 hours and the dialysate was changed every 12 hours. The purified enzyme was used for enzyme activity determination. Crude enzymes were also extracted in vivo from different parts of jujube diseased fruit inoculated with spore suspension. The tissues were cut into slices and put into mortar, grinded with sodium chloride at 4 ℃, centrifuged at 4 ℃ and 5 000 r·min~(-1) after filtration, and the supernatant was collected as the crude enzyme for later use. Activities of six cell wall degrading enzymes were measured using the 3, 5-dinitrosalicylic acid(DNS) method and the Coomassie blue staining method. Among them, the activities of polygalacturonase, carboxymethylcellulase, β-glucosidase,xylanase and polygalacturonase were measured by the 3, 5-dinitrosalicylic acid method using pectin,carboxymethyl cellulose, salicin and xylan as substrate, respectively. Substrate solution was prepared with citric acid buffer. Using citric acid buffer solution as the blank, the enzymic hydrolysis was carried out at 50 ℃ for 30 to 60 minutes. The absorbance of the reaction mixture was measured by UV-Vis spectrophotometer, and the the enzyme activities were calculated according to the reducing sugar released. The pectin methyl trans-eliminating enzyme was determined by Coomassie Brilliant Blue method. The color density of the protein-Coomassie brilliant blue complex solution was proportional to protein concentration. The differences in color of the solution created by enzymic reactions was used to calculate the cell wall degrading enzyme activity.【Results】Jujube black spot bacteria produced six cell wall degrading enzymes under the induction of different carbon sources. They were polygalacturonase,carboxymethylcellulase, β-glucosidase, xylanase, polymethylgalacturonase and pectin methyl transeliminase. However, there were some differences in activity among enzymes under different inducers.When the jujube pulp was used as the inducer, the activity of β-glucosidase produced by the black spot pathogen was significantly higher than that of the other five enzymes, indicating that β-glucosidase played an important role in the pathogenesis of black spot pathogen. Filter paper as the inducer also produced the highest β-glucosidase activity. The activities of cell wall degrading enzymes produced by jujube black spot in infected jujube fruit was consistent with the results induced by different carbon sources in vitro, with the activity of β-glucosidase being higher than the other five enzymes. Moreover, the activity of β-glucosidase at the junction of diseased and healthy parts was the highest among different tissues sites.【Conclusion】Different inducers have different effects on the cell wall degrading enzymes secreted by the black spot pathogen, and the inducer most favorable for pathogen growth has an important effect on the enzyme production. Filter paper can be used as a good inducer of β-glucosidase. Combining the enzyme activity assay results in vitro and in vivo, it is speculated that β-glucosidase is the major cell wall degrading enzymes that play a role in the pathogenesis of the black spot pathogen. The key part of the impact is at the edge of the diseased tissue.
引文
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[2]许瑛,姚兆群,王兰,谢建明,赵思峰.阿拉尔地区枣园不同时期枣花、枣果黑斑病菌带菌量检测[J].新疆农业学,2017,54(10):1911-1919.XU Ying,YAO Zhaoqun,WANG Lan,XIE Jianming,ZHAO Sifeng. Detection of the amount of Fungi in the different infection periods of jujube Black Spot Disease in Alar Area[J]. Xinjiang Agricultural Sciences,2017,54(10):1911-1919.
[3]靳增军.冬枣黑疔病病原鉴定、生物学特性研究及有效药剂筛选[D].保定:河北农业大学,2006.JIN Zengjun. Identification,Bio-characters and chemical screening of pathogens causing black spot“Dongzao”Jujube[D]. Baoding:Hebei Agricultural University,2006.
[4]李凤琴.链格孢毒素及其食品卫生问题(综述)[J].中国食品卫生杂志,2001(6):45-49.LI Fengqin. Alternaria toxin and its food hygiene problems(a summary)[J]. Chinese Journal of Food Hygiene,2001(6):45-49.
[5]宋博,徐兵强,朱晓锋,阿布都克尤木·卡德尔,杨森.南疆红枣黑斑病病原鉴定及其保守基因序列分析[J].植物病理学报,2017,47(3):411-415.SONG Bo,XU Bingqiang,ZHU Xiaofeng,Abudu Keyimu·Kader,YANG Sen. Identification of jujube black spot pathogens in Southern Xinjiang and its sequence analysis of conserved genes[J]. Acta Paytopathologica Sinica,2017,47(3):411-415.
[6]包慧芳,王宁,侯敏.新疆红枣黑斑病病原菌鉴定及拮抗细菌筛选[J].新疆农业科学,2016,53(9):1667-1673.BAO Huifang,WANG Ning,HOU Min. Identifica-tion of the pathogen and screening of the antagonistic bacteria of Jujube Black Spot Disease in Xinjiang[J]. Xinjiang Agricultural Sciences,2016,53(9):1667-1673.
[7]马荣,刘晓琳,梁英梅,闫军,陈宝军,赵景玲,张涛.枣果黑斑病发生相关因素分析及田间药效试验[J].中国农学通报,2015,31(16):182-189.MA Rong,LIU Xiaolin,LIANG Yingmei,YAN Jun,CHEN Baojun,ZHAO Jingling,ZHANG Tao. Analysis of the influencing factors on disease occurrence of jujube black spot and field efficacy test[J]. Chinese Agricultural Science Bulletin,2015,31(16):182-189.
[8]张大智,詹儒林,柳凤,李国平,赵艳龙,常金梅.杧果细菌性角斑病菌细胞壁降解酶的致病作用[J].果树学报,2016,33(5):585-593.ZHANG Dazhi,ZHAN Rulin,LIU Feng,LI Guoping,ZHAO Yanlong,CHANG Jinmei. Pathogenic effect of cel-l wall degrading enzymes produced by pathogen causing mango bacterial leaf spot[J]. Journal of Fruit Science,2016,33(5):585-593.
[9]陈捷,唐朝荣,邹庆道,宋佐衡.玉米纹枯病菌致病因子的研究[J].吉林农业大学学报,1998(增刊):138.CHEN Jie,TANG Zhaorong,ZOU Qingdao,SONG Zuoheng.On pathogenocity factors in Corn Sheath Blight[J]. Journal of Jilin Agricultural University,1998(Suppl.):138.
[10] SIAH A,DEWEER C,DUYMEC F,SANSSENéJ,DURAND R,HALAMA P,REIGNAULT P. Correlation of in planta endobeta-1,4-xylanase activity with the n-ecrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola[J]. Plant Pathology,2010,59:661-670.
[11]李宝聚,周长力,赵奎华,李凤云,陈红漫.黄瓜黑星病菌致病机理的研究Ⅱ细胞壁降解酶及其在致病中的作用[J].植物病理学报,2000(1):13-18.LI Baoju,ZHOU Changli,ZHAO Kuihua,LI Fengyun,CHEN Hongman. Pathogenic mechanism of scab of cu-cumber caused by Cladosporium cucumerinumⅡthe cell wall degrading enzymes and its pathogenic action[J]. Acta Paytopathologica Sinica,2000(1):13-18.
[12]陈晓林,牛程旺,李保华,李桂舫,王彩霞.苹果树腐烂病菌产生细胞壁降解酶的种类及其活性分析[J].华北农学报,2012,27(2):207-212.CHEN Xiaolin,NIU Chengwang,LI Baohua,LI Guifang,WANG Caixia. The kinds and activities of cell wall-degrading enzymes produced by Valsa ceratosperma[J]. Acta Agriculturae Boreali-Sinica,2012,27(2):207-212.
[13]董宁,冯宏祖,王兰,纪显洪,于丝丝.南疆骏枣黑斑病症状表现及病原菌鉴定[J].植物保护学报,2016,43(6):922-927.DONG Ning,FENG Hongzu,WANG Lan,JI Xianhong,YU Sisi. The disease symptom and the pathogen identification of jujube black spotin southern Xinjiang[J]. Journal of Plant Protection,2016,43(6):922-927.
[14] DOUAIHER M N,NOWAK E,DURAND R,HALAMA P,REIGNAULT P. Correlative analysis of Mycosphaerella graminicola pathogenicity and cell wall degrading enzymes produced in vitro:the importance of xylanases and poly-galacturonases[J]. Plant Pathology,2017,56:79-86.
[15]董章勇,王振中.植物病原真菌细胞壁降解酶的研究进展[J].湖北农业科学,2012,51(21):4697-4700.DONG Zhangyong,WANG Zhenzhong. Research progress of fungal cell wall-degrading enzyme[J]. Hubei Agricultural Sciences,2012,51(21):4697-4700.
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